collecting macromolecular crystallographic data at synchrotrons andrew howard aca summer school 12...
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Collecting Macromolecular Crystallographic Data at
Synchrotrons
Andrew HowardACA Summer School12 July 2007
Synchrotrons are useful, not just fashionable
• You can do almost any experiment better and faster at a storage ring than in a conventional lab; and there are experiments that you can only do at a storage ring.
What we need to think about
• Why synchrotrons help: Factors, parameters
• How they make things harder
• How synchrotron data collection is different from domestic data collection
• How macromolecular crystallography is different from other storage-ring apps
How synchrotrons help
• Fluence
• Brilliance
• Tunability
• Collimation
• Resources
• 1013 Xph/s/mm2
• 1017 Xph/s/mm2/mrad2
• E = 12398.0 ± 0.4eV
• FWHM(v) < 100 µm
• Lasers, experts, labs …
Some definitions and unitsQuantity Definition Units Value
Flux # photons / Xph/ 1012
unit time sec
Fluence flux / unit area (Xph/sec)/ 1013
mm2
Brilliance fluence/ Xph/sec/ 1017
solid angle* (mm2-mrad2)
Brightness flux/solid angle* Xph/sec/ 1016
mrad2
* Sometimes defined in terms of bandwidth, e.g.brilliance = (fluence/solid angle)/bandwidth
Which parameters really matter?
• For most macromolecular crystallographic experiments fluence is the relevant parameter: we want lots of photons entrained upon a small area
• Brilliance matters with very large unit cells where a high divergence is bad
What does high fluence do?
• Allows us to get good signal-to-noise from small samples
• Allows us to irradiate segments of larger samples to counteract decay
• Many experiments per day
• Allows us to contemplate experiments we would never consider with lower fluence
What does high brilliance do?
• How do we separate spotsif the unit cell length > 500 Å?
– Back up the detector
– Use tiny beams
• Large beam divergence will prevent either of those tools from working
Tunability
• Monochromatic experiments:We’re allowed to choose the energy that works best for our experiment
• Optimized-anomalous experiments:We can collect F(h,k,l) and F(-h,-k,-l) at the energy where they’re most different
• Multiwavelength:pick 3-4 energies based on XAS scan and collect diffraction data at all of them
What energies are available?
• Depends on the storage ring
• Undulators at big 3rd-generation sources:3-80 KeV
• Protein experiments mostly 5-25 KeV– Below 5: absorption by sample & medium
– Above 25: Edges are ugly, pattern too crowded
• Some beamlines still monochromatic
Energy resolution &spectral width
• Energy resolution: how selective we can reproducibly produce a given energy– Typically ~ 0.4 eV at 3rd-Gen sources
– Need: E < [Epeak - Eedge (Se)] ~ 1.4 eV
• Spectral width: how wide the energy output is with the monochromator set to a particular value
Collimation
• Everyone collimates. What’s special?
– Beam inherently undivergent
– Facility set up to spend serious money making collimation work right
• Result: we can match the beam size to the crystal or to a desired segment of it
ResourcesStorage rings are large facilities with a number
of resources in the vicinity
• Specialized scientific equipment (lasers)
• Smart, innovative people
• Sometimes: well-equipped local labs where you can do specialized sample preparations
Why wouldn’t we do this?
• Beamtime is still scarce
• You’re away from your home resources
• Disruption of human schedules
– Travel
– 24-hour to 48-hour nonstop efforts
– Bad food
• Extra paperwork:Safety, facility security, statistics
How does synchrotron crystallography differ from lab crystallography?• Time scale very foreshortened
• Multiwavelength means new experimental regimes
• Distinct need for planning and prioritizing experiments
• Robotics: taking hold faster @ beamlines
How does macromolecular crystallography differ from other beamline activities?• “Physics and chemistry groups at the beamline
do experiments;crystallographers do data collection”
• Expectation: zero or minimal down-time between users
• Often: well-integrated process from sample mounting through structure determination
Where will we collect data?
• SER-CAT: 22-ID
• SBC-CAT: 19-BM
• GM/CA-CAT: 23-ID
• NE-CAT: 8-BM (perhaps)
• DND-CAT: 5-ID
• BioCARS: 14-BM-C
Southeast Regional CAT (22)• Established ~2002
• Run as an academic consortium of about 25 universities, mostly in the southeast, with some legislative or provost-level support
• 30% of my salary from there!
GM/CA-CAT (23)
• Established around 2004 as a site for NIH GM and Cancer grantees, particularly those working on structural genomics and cancer therapeutics
• First APS facility to build out multiple endstations on an insertion device line that are capable of simultaneous use
BioCARS• Established around 1997 to do cutting-
edge crystallographic projects, particularly involving time-resolved techniques and BSL-2 or BSL-3 samples
SBC-CAT
• Oldest macromolecular crystallography facility at the APS
• 19-ID: more structures solved than any other beamline in the world
• Good for all sizes and resolutions